Staining techniques

Staining techniques
• Staining is coloring with dye to make some structures more visible.
• Staining is used to increase visibility as most bacterial are transparent
and difficult to see under light microscope.
• Before microorganisms can be stained, they must be fixed or attached
to the microscopic slide; otherwise they might be washed a way by the
dye.
• For fixing smear preparation is needed.
• Smear: A thin film of a solution of microbes on a slide.
• A smear is usually fixed to attach the microbes to the slide and to kill
the microbes.
 Wet mount versus stained smear
 Wet mount
• Cell suspended in fluid a drop or two of the culture is then placed on
a slide and overlaid with a cover glass
• Cove glass can damage larger cells and might dry or contaminate the
observers fingers
 Smear preparation
• A smear is a thin film of bacterial suspension prepared on a slide for
microscopic examination
• It is prepared by spreading the material (suspension) over the surface
of a slide and allowing it to air dry.
• The specimen is fixed ether by heat or chemicals (ethanol,
formaldehyde, etc) after the smear is air dries.
• The stain is applied and then washed off with water .
• The slide is then blotted with absorbent paper.

• The stained microorganisms are now ready for microscopic

examination.
 Stains (dyes)
• Chemically, dyes are usually salt, although a few are bases or acids
composed of a positive and a negative ion, one of which is colored and is
called the chromophore (the part of molecule that is responding for
color).
• Dyes can be basic, acidic or neutral.

• The color of basic dyes is in the positive ion e.g. methylene blue , crystal
violet, basic fuchsine, malachite green and safranin are positive charged.
• Positive charged dyes react with /bind to the negatively charge nucleic
acid and proteins inside cell and to polysaccharides on the bacterial cell
surface.
• In acidic dye (negatively charged), the color is in the negative ion.

• They are not attached by most bacteria because the negative ions

are repelled by the negatively charged bacterial surface.

• Negatively charged dyes stain the background instead of the cell.

This is called negative staining(preparation of colorless bacteria

against a colored background) .

• They are important in the observation of overall cell shapes, size

and capsule. Example of acidic dyes include eosin, nigrosin,

indian ink, rose Bengal and acid fuchsin.

• Neutral dyes are made by combining acidic and basic dyes.

• The colored part is contained in both positive and negative ions

(e.g. Giemsa stain).
• They are most useful for staining complex cells of higher forms
because they permit differentiation of interior structures, some
of which are basic and some are acidic.
• They are used to stain nucleic acids and cytoplasm.
 Types of staining techniques

• There are two types of categories of staining techniques: simple

and differential staining.
A) simple staining
• Simple staining employs a single dye, most commonly methylene
blue, crystal violet, basic fuchsin and safranin.
• It generally stains the bacterial cell the same color.
• Simple staining is used to reveal size, shape and arrangement of
bacteria .
• There are two categories of simple staining.
B) differential staining

• Differential staining used more than one dye and is used to

distinguish between structure within a cell or between types of

bacteria by staining them different colors.

• Gram staining, acid-fast staining, flagella, capsule and endospore

staining are examples of differential staining. However, the first

two are the most frequently used.

 Staining Principles

• Acidic (-ve) / Basic (+ve)

• Simple Stains

• Differential Stains

• Special Stains
 C) Gram Stain
• Differential Stain
– 2 colors for Gram +ve and Gram –ve
– Developed by Hans Gram
– Distinguishes microbes based on peptidoglycan content in cell wall
• In the first step the smear is stained with basic dye crystal violet
(Primary stain) followed by treatment with iodine solution functioning
as mordant.
• CV-I complex
– Crystal violet + iodine forms a large molecular complex
• Decolorized Alcohol wash
• Cannot wash out of the Gram +ve cell wall
• Gram –ve wall is easily disrupted by alcohol wash
• Saffranin counterstain
• Gram +ve cells stay purple
• Gram –ve cells stain pink
• Diagnostic importance
– Peptidoglycan is a target for many antibiotics when present in
a thick layer
• Penicillins
• Monobactams
• Carbapenems
• Iodine increases the interaction between cell & dye so that cell

stains strongly.

• The smear is next decolorized by washing with ethanol or acetone.

• Finally smear is counter-stained with a simple basic dye different in

color from Crystal violet.

• Safranin is the most common counter stain which colours Gram

negative bacteria pink to red and leaves Gram positive bacteria dark

purple.
 Appearance After Each Step

1. Crystal violet

2. Addition of Iodine

3. Alcohol rinse step

4. Safranin Counterstain
 D)Ziehl-Neelsen Acid Fast Stain
• Acid Fast Bacteria
• Mycobacterium genus
identification
• These bacteria have cell wall with
high lipid content such as mycolic
acid .
• A group of branched chain
hydroxy lipids,
• which prevent dyes from readily
binding to cells.
• They can be stained by Ziehl-Nulsen method, which uses heat and

phenol to derive basic fuchsin into the cells.

– Mycrobacterium spp. were penetrated with basic fuchsin, not
easily decolourized by acidified alcohol (acid alcohol) and thus
are said to be acid fast.
– Genus is one of few that stain pink

– Diagnosis
• Tuberculosis

• Leprosy
 E) Negative (Capsule) Stain
• Visualizes the Capsule
– Thick glycocalyx around
cell
– Negatively charged dyes
• Repelled by –ve
charge on glycocalyx
• Halo effect is seen
– Cells are counterstained
with positively charged
dye
• Virulence factor
– Repels phagocytic WBC
– May prevent antibiotic
entry into cells
 F) Endospore Stain (Schaefer-Fulton)
• Endospores are hard to see
with Gram stain
– Waxy dipicolinic acid
• Visualizes pathogens
– Clostridium tetani
– C. botulinum
– C. perfringens
– Bacillus anthracis
• Vegetative cells vs.
Endospores
– Sterilization challenge
– Heat and dessication-
resisitant
• Spore formation takes place in some bacterial genera to
withstand unfavourable conditions.
• All bacteria cannot form spores, only few bacterial genera
including Bacillus and Clostridium,
 It produce sporulating structure inside vegetative cells called
endospore.

• Endospore morphology and location vary with species and are

valuable for identification.
• Endospores are not stained well by most dyes, but once
stained, they strongly resist decolorization.
• In the Schaffer-Fulton procedure, endospores are first stained by

heating bacteria with malachite green,

 which is very strong stain that can penetrate endospores.

• After malachite green treatment, the rest of the cell is washed free

of dye with water and is counter-stained with safranin.

• This technique yields a green endospore with red vegetative cell.

Special Stains See Fig 3.14

• Endospore stain: Heat is required to drive a

stain into the endospore.

• Flagella staining: requires a mordant to make

the flagella wide enough to see.
• Capsule stain uses basic stain and negative
stain
Review of different staining techniques
Important Staining Reactions in Microbiology

For Gram stain

technique compare to
Fig 3-12
 CHAPTER THREE

Taxonomy Of Microbes(Major groups

of microorganisms)